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• W2908799316 abstract "Article14 January 2019Open Access Transparent process An increase in neural stem cells and olfactory bulb adult neurogenesis improves discrimination of highly similar odorants Sara Bragado Alonso Sara Bragado Alonso CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Janine K Reinert Janine K Reinert Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nicolas Marichal Nicolas Marichal Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK Search for more papers by this author Simone Massalini Simone Massalini CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Benedikt Berninger Benedikt Berninger orcid.org/0000-0003-2652-2782 Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK Search for more papers by this author Thomas Kuner Thomas Kuner Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Federico Calegari Corresponding Author Federico Calegari [email protected] orcid.org/0000-0002-3703-2802 CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Sara Bragado Alonso Sara Bragado Alonso CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Janine K Reinert Janine K Reinert Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Nicolas Marichal Nicolas Marichal Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK Search for more papers by this author Simone Massalini Simone Massalini CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Benedikt Berninger Benedikt Berninger orcid.org/0000-0003-2652-2782 Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK Search for more papers by this author Thomas Kuner Thomas Kuner Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Federico Calegari Corresponding Author Federico Calegari [email protected] orcid.org/0000-0002-3703-2802 CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany Search for more papers by this author Author Information Sara Bragado Alonso1, Janine K Reinert2, Nicolas Marichal3,4, Simone Massalini1, Benedikt Berninger3,4, Thomas Kuner2 and Federico Calegari *,1 1CRTD Center for Regenerative Therapies Dresden, School of Medicine, TU Dresden, Dresden, Germany 2Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany 3Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany 4Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK *Corresponding author. Tel: +49 3514 5882204; E-mail: [email protected] The EMBO Journal (2019)38:e98791https://doi.org/10.15252/embj.201798791 See also: M Lipovsek & MS Grubb (March 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Adult neurogenesis is involved in cognitive performance but studies that manipulated this process to improve brain function are scarce. Here, we characterized a genetic mouse model in which neural stem cells (NSC) of the subventricular zone (SVZ) were temporarily expanded by conditional expression of the cell cycle regulators Cdk4/cyclinD1, thus increasing neurogenesis. We found that supernumerary neurons matured and integrated in the olfactory bulb similarly to physiologically generated newborn neurons displaying a correct expression of molecular markers, morphology and electrophysiological activity. Olfactory performance upon increased neurogenesis was unchanged when mice were tested on relatively easy tasks using distinct odor stimuli. In contrast, intriguingly, increasing neurogenesis improved the discrimination ability of mice when challenged with a difficult task using mixtures of highly similar odorants. Together, our study provides a mammalian model to control the expansion of somatic stem cells that can in principle be applied to any tissue for basic research and models of therapy. By applying this to NSC of the SVZ, we highlighted the importance of adult neurogenesis to specifically improve performance in a challenging olfactory task. Synopsis A transitory expansion of neural stem cells results in an increase in adult born neurons that functionally integrate in the mouse olfactory bulb. These neurons improve the performance of challenging olfactory tasks by increasing the discrimination of similar odorants. A novel mouse model for the inducible and tissue-specific expression of Cdk4/cyclinD1 (4D) allows the expansion of somatic stem cells. Expansion of neural stem cells in the adult subventricular zone triggers the long-term increase in adult neurogenesis. Supernumerary neurons can physiologically migrate, mature and integrate in the olfactory bulb circuitry. Mice with increased adult neurogenesis are better specifically at discriminating highly similar odorants. Our mouse model might provide an important tool toward investigating the use of somatic stem cells for recovery of organ function in disease models. Introduction The subventricular zone (SVZ) of the lateral ventricles is the primary neurogenic niche of the adult mammalian brain harboring neural stem cells (NSC) throughout life. This endogenous source of new neurons holds great potential toward therapy, and major efforts are aimed to understand the mechanisms governing the proliferation and differentiation of NSC and the role of adult neurogenesis in cognitive performance and brain plasticity (Silva-Vargas et al, 2013; Lim & Alvarez-Buylla, 2014; Lepousez et al, 2015; Sailor et al, 2017). Specifically, slowly dividing progenitors of embryonic origin (Fuentealba et al, 2015; Furutachi et al, 2015) become activated NSC (B1 cells) of the adult SVZ and give rise to intermediate progenitors (C cells) that produce neuroblasts (A cells) migrating through the rostral migratory stream (RMS) and generating interneurons of the olfactory bulb (OB; Doetsch et al, 1997). As a result, newborn granule and periglomerular cells are continuously added to the OB, contributing to the plasticity of the local circuitry throughout life by modulating the activity of mitral and tufted cells whose output is projected to the olfactory cortex (Hack et al, 2005; Mizrahi et al, 2006; Brill et al, 2009; Ghosh et al, 2011; Miyamichi et al, 2011). Studies attempting to link neurogenesis with olfaction yielded conflicting results pointing to a yet unresolved debate about the role of newborn neurons. For instance, depleting NSC decreased olfactory memory with some (Sakamoto et al, 2014) or no (Breton-Provencher et al, 2009; Lazarini et al, 2009) effect on odor discrimination or learning. Similarly, impairing the migration or survival of newborn neurons had either no effect (Kim et al, 2007) or inhibited odor discrimination (Gheusi et al, 2000; Bath et al, 2008). Seemingly contradicting these findings, promoting neuronal survival also decreased odor discrimination (Mouret et al, 2009) or, alternatively, improved learning (Wang et al, 2015). Moreover, activating newly integrated cells was reported to facilitate learning and memory by some studies (Alonso et al, 2012; Gschwend et al, 2015), while, conversely, others showed that enhancing the inhibitory activity of all granule cells in the OB improved odor discrimination time but not learning or memory (Abraham et al, 2010; Nunes & Kuner, 2015). These inconsistencies are likely due to the very different approaches previously used by which the intrinsic properties of the mature and/or newborn neurons themselves have been manipulated including at the level of their migration, survival, integration, or electrophysiological properties. In fact, studies investigating whether a specific expansion of NSC resulting in an increased number of otherwise physiologically normal, unmanipulated neurons improves brain function are lacking. Addressing this question is fundamental to identify the specific role of adult neurogenesis in olfaction and explore systems to expand NSC, and perhaps also other somatic stem cells, for therapy. Our and other groups have shown that the cell cycle regulators Cdk4/cyclinD1 (4D) can be used to regulate the expansion not only of NSC (Lange et al, 2009; Artegiani et al, 2011; Nonaka-Kinoshita et al, 2013) but also human hematopoietic (Mende et al, 2015) and pancreatic β-cell (Azzarelli et al, 2017; Krentz et al, 2017) precursors. Hence, here we decided to develop a versatile transgenic mouse model to temporarily control 4D in any tissue of choice. We then used this tool to assess the effects of a cell-intrinsic expansion of adult NSC without a manipulation of their niche and resulting in the increased generation of physiologically normal neurons to study their role in olfactory performance. Results Temporal control of NSC expansion in the SVZ Our group has shown that a transient 4D overexpression promoted the proliferation of NSC by shortening their cell cycle, specifically G1. Concomitantly, a shortening of G1 promoted a switch of NSC fate from differentiative to proliferative divisions resulting in the subsequent increase in the number of newborn neurons generated during embryonic cortical development and adult hippocampal neurogenesis (Lange et al, 2009; Artegiani et al, 2011; Nonaka-Kinoshita et al, 2013). Therefore, in order to investigate the effects of increased neurogenesis in odor discrimination, we generated a triple transgenic mouse line by crossing nestinCreERT2 (Imayoshi et al, 2008), ROSA26rtTA-flox (Belteki et al, 2005), and tet4D-RFP (Nonaka-Kinoshita et al, 2013) mice (Fig 1A). This system was designed to allow the tamoxifen (Tam)-dependent activation of rtTA specifically in NSC followed by an inducible and reversible 4D expression, together with RFP as reporter, in a doxycycline (Dox), time-dependent manner. Notably, the ROSA26rtTA-flox/tet4D-RFP line would also allow 4D to be in principle controlled in any other tissue of choice by simply crossing this line with any appropriate Cre driver mouse. Figure 1. Transgenic model and effects of acute 4D overexpression on NSC and progenitors of the SVZ Drawings of the nestinCreERT2 (Imayoshi et al, 2008), ROSA26rtTA-flox (Belteki et al, 2005), and tet4D-RFP (Nonaka-Kinoshita et al, 2013) alleles of the 4D line. From top to bottom: experimental design of 4D induction, fluorescence pictures of the SVZ of a 4D+ mouse and quantification of the proportion of RFP− (black) and RFP+ (red) progenitors among B1, C, and A cells identified with markers as indicated. From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D− (left) and 4D+ (right and insets magnified) mice, and quantification of the proportion of BrdU+ among B1, C, and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars). Quantification of the absolute number of B1, C, and A cells (identified as in B) in the SVZ of 4D− (white) and 4D+ (black) mice regardless of RFP expression (RFP+/−). Data information: (B–D) Mean ± SEM; *P < 0.05 and **P < 0.01 calculated by unpaired Student's t-test; N = 3 mice, n > 423 cells for each quantification. Scale bar = 50 μm (B and C) or 20 μm (inset in C). Download figure Download PowerPoint Triple homozygous, adult nestinCreERT2+/+/ROSA26rtTA-flox+/+/tet4D-RFP+/+ mice (referred to as 4D+; see Materials and Methods and Appendix) for the strategy used to obtain this line) were administered Tam for 3 days followed by 4 days of clearance and the subsequent start of Dox administration (defined to as day 0; Fig 1B). After 4 days of Dox, both RFP mRNA and endogenous fluorescence were detected along the SVZ and RMS that, as expected, still did not reach the OB (Fig EV1A). In contrast, neither RFP mRNA nor protein were detectable by in situ hybridization or antibody enhancement, respectively, in any other brain area including the hippocampus (Fig EV1A and A’), which is likely due to the lower dosage of Tam relative to that optimized for this niche (Imayoshi et al, 2008; Artegiani et al, 2011). No RFP protein could be detected either in the olfactory epithelium (Fig EV1A) underlying the SVZ-specific expression of 4D despite the presence of nestin+ cells in other regions of the nervous system. Click here to expand this figure. Figure EV1. Characterization of the transgenic model and effect of 4D on the RMS A. Fluorescence image of a sagittal section of a 4D+ brain after a 4-day treatment with doxycycline showing RFP signal confined to the SVZ and RMS (nuclei counterstained with DAPI; blue). Insets show representative images of specific brain regions (as indicated) and the olfactory epithelium. A’. Phase contrast picture of the SVZ upon in situ hybridization against mRNA for RFP in a 4D+ brain treated as in (A) and sacrificed immediately after (left) or 2 days after (right) doxycycline administration. B, C. Experimental design (top), fluorescence pictures (left with magnified insets), and quantifications (right) of BrdU incorporation in the RMS (B) or SVZ (C). (B) shows the proportion of BrdU in C (Mash1+) and A (DCX+) cells in 4D− (white) and 4D+ (red; among RFP+) mice. (C) shows the proportion of RFP− (black) and RFP+ (red) among BrdU+ cells of 4D+ mice. (A) OB, olfactory bulb; RMS, rostral migratory stream; LV, lateral ventricle; DG, dentate gyrus; OE, olfactory epithelium. (A–C) Tam, tamoxifen; Dox, doxycycline. (B, C) Mean ± SEM; **P < 0.01; unpaired Student's t-test; N = 3 mice and n > 1,100 cells. Scale bars = 500 (A top, A’), 100 (insets A and A’), 50 (B and C), and 20 (insets B and C) μm. Download figure Download PowerPoint Within the SVZ, 4D-RFP induction occurred to a similar degree in C and A progenitor cells (72.1 ± 4.8 and 68.2 ± 3.2% of all Mash1+ and DCX+, respectively) and to a lesser extent in activated B1 cells identified as either EGFR+Mash1− or nestin+S100β− (Codega et al, 2014; 54.4 ± 6.1 and 55.1 ± 2.8%, respectively; Fig 1B). Moreover, RFP mRNA levels were back to undetectable levels 2 days after withdrawing Dox (Fig EV1A’), evidencing the efficiency of our on/off expression system and lack of leakiness. We next investigated the effects of a 4-day 4D overexpression on proliferation by one pulse of BrdU 12 h before sacrifice (Fig 1C). Hereafter, triple homozygous, nestinCreERT2+/+/ROSA26rtTA-flox+/+/tet4D-RFP−/− mice (referred to as 4D−) in the same genetic background of 4D+ mice and equally treated with Tam and Dox were used as negative controls. First, we quantified the proportion of BrdU+ cells among activated B1, C, and A cells in 4D− and 4D+ mice. We found that in 4D+ mice the vast majority (> 80%) of RFP+ cells was also BrdU+ (Fig 1C). Consistently, the proportion of BrdU+ cells among activated B1 cells had substantially increased relative to 4D− mice (from 6.0 ± 0.3 to 42.9 ± 5.9%, P < 0.005 and from 28.6 ± 2.1 to 47.7 ± 1.7%, P < 0.005, among EGFR+Mash1− and nestin+S100β− cells, respectively). Note that the different fold-increase by the use of the two marker pairs is likely due to the reported degradation of nestin in S/G2 (Sunabori et al, 2008; Codega et al, 2014) during which BrdU is incorporated; Fig 1C). A similar increase in the proportion of BrdU+ cells was also found among C and A cells in both the SVZ (from 46.9 ± 2.3 to 76.1 ± 5.4%, P < 0.01 and from 35.8 ± 3.2 to 69.5 ± 6.4%, P < 0.01, for Mash1+ and DCX+ cells, respectively; Fig 1C) and RMS (from 47.9 ± 2.0 to 78.5 ± 2.1%, P < 0.01 and from 39.0 ± 10.5 to 65.4 ± 8.8%, P = 0.13, for Mash1+ and DCX+ cells, respectively; Fig EV1B). These data were consistent with the known effect of 4D in shortening G1 (Lange et al, 2009; Artegiani et al, 2011; Nonaka-Kinoshita et al, 2013) and underlying the observed increase in BrdU incorporation. Yet, despite the massive increase in BrdU+ cells, these results were hard to interpret given that RFP+ cells in 4D+ mice represented only a fraction (ca. 50%) of all stem and progenitor cells (Fig 1B). Hence, comparison of this subpopulation of RFP+ cells in 4D+ mice with all cells in 4D− mice might have resulted in a bias if RFP expression was to be enriched in fast-proliferating cells. This was unlikely because BrdU labeling in 4D+ mice prior to the beginning of Dox administration, i.e., before a phenotype could be triggered, led to a similar proportion of BrdU+ cells among the RFP− and RFP+ population (Fig EV1C) indicating that 4D-RFP induction did not bias toward fast-proliferating cells. Nevertheless, to directly and incontrovertibly exclude the effects of any potential bias in 4D-RFP expression, we quantified the absolute number of BrdU+ cells in 4D+ and 4D− mice regardless of RFP expression. This was also important because the previous increase in the proportion of BrdU+ cells (Fig 1C) primarily reflected a change in cell cycle parameters, but not necessarily in fate, of neural stem and progenitor cells, which could only be proven by observing an increase in their numbers irrespective of BrdU incorporation. We found that a 4-day induction of 4D triggered an increase by 30% in activated B1 cells per tissue volume (from 3.3 ± 0.2 × 104 to 4.4 ± 0.3 × 104, P < 0.05, and from 51.2 ± 1.4 × 104 to 66.6 ± 4.3 × 104, P < 0.05, EGFR+Mash1− and nestin+S100β− cells/mm3, respectively) and 15% in C cells (from 43.9 ± 1.6 × 104 to 52.7 ± 1.2 × 104 Mash1+ cells/mm3, P < 0.01) throughout the SVZ (Fig 1D). This increase in cell numbers in 4D+ mice occurred without distinguishing between RFP− and RFP+ cells indicating that the real 4D-triggered effect on cell fate was greater, in principle the double, than the one assessed. Altogether, the observed increase in the proportion of BrdU incorporation and number of NSC supports the notion (Lange & Calegari, 2010; Borrell & Calegari, 2014) that 4D overexpression not only induces a faster cell cycle in NSC but also changes their fate by promoting proliferative divisions and expansion of their pool over time. 4D expands NSC without inducing their depletion and increases neurogenesis We next addressed the long-term effect of an acute 4D overexpression. In particular, we investigated whether (i) enhanced NSC proliferation was reversible upon turning off 4D, thus allowing their switch to differentiation; (ii) supernumerary NSC could re-enter quiescence, which is essential to prevent their long-term depletion; (iii) the balance between gliogenic vs. neurogenic commitment was maintained without altering NSC multipotency; and finally, (iv) expansion of NSC increased neurogenesis without compensatory effects. To address all these questions, we designed a common experimental paradigm by which 4D was induced for 4 days concomitantly with BrdU administration followed by a 30- or 60-day chase without Dox (Figs 2A and EV2A). Figure 2. Chronic effect of 4D overexpression on NSC and OB neurogenesis A. Experimental design used to assess the chronic effect of a transient 4D induction with BrdU and EdU given during Dox administration or 1 h before sacrifice, respectively. B–E. From top to bottom: fluorescence pictures of the SVZ (B–D) or OB (E) and absolute number (B, C, and E) or proportions (C–E) of cells in 4D− (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Insets in (C) are magnified (right) with arrowheads pointing label-retaining NSC (white) or astrocytes (empty). Arrowheads in (D) point cell doublets (among RFP+ protein-retaining cells in 4D+). (E) GL, glomerular; EPL, external plexiform; MCL, mitral cell and GCL, granule cell layers. Data information: (B–E) Mean ± SEM; *P < 0.05, **P < 0.01 assessed by unpaired Student's t-test (bar graphs) or Fisher's exact test (pie graphs); N = 3 mice, n > 285 cells for each quantification. Scale bars = 50 μm (B, C, and E) and 20 μm (D and insets in C). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Long-term effect of 4D overexpression on NSC and OB neurogenesis A. Experimental paradigm to investigate long-term effects of 4D expression. Tam, tamoxifen; Dox, doxycycline. B, C. From left to right: fluorescence pictures of the SVZ (B) or OB (C) and number per mm3 (bar graphs) or proportions (pie graphs), of cells in 4D− (left pictures and white bars) or 4D+ (right pictures and black bars) mice and scored positive for markers as indicated. Insets in (B) are magnified (right) with arrowheads pointing label-retaining NSC (white) or astrocytes (empty). (C) GL, glomerular; EPL, external plexiform; MCL, mitral cell; and GCL, granule cell layers. (B, C) Mean ± SEM; *P < 0.05, **P < 0.01; unpaired Student's t-test or Fisher's exact test (pie graphs); N = 4 mice, n > 210 cells. Scale bars = 50 (B and C) and 20 (caption in B) μm. Download figure Download PowerPoint First, at the end of the described treatment, a single pulse of EdU was given 1 h before sacrifice to investigate whether the 4D effect on the cell cycle and proliferation was reversible (Fig 2A). No difference was found in the number of EdU+ cells (31.4 ± 3.2 × 104 and 33.2 ± 1.7 × 104 EdU+ cells/mm3 in 4D− and 4D+ mice, respectively, P = 0.66), indicating that the 4D effect was fully reversible and proliferation was restored back to physiological levels (Fig 2B). Second, BrdU label retention was used to address entry into quiescence (Doetsch et al, 1999). NSC that were cycling during Dox administration and retained the label following a 30-day chase were identified as BrdU+Sox2+S100β− (Fig 2C; white arrowheads) and found to have increased by twofold in 4D+ mice relative to control (1.9 ± 0.3 × 104 vs. 3.8 ± 0.3 × 104 cells/mm3, P < 0.01; Fig 2C). This twofold increase in long-term, label-retaining cells seemingly persisted even after a 60-day chase despite the overall age-dependent decrease in the number of quiescent NSC in both cohorts of mice (1.1 ± 0.1 × 104 vs. 1.8 ± 0.3 × 104 BrdU+Sox2+S100β− cells/mm3 in 4D− and 4D+, respectively, P = 0.09; Fig EV2A and B), suggesting a long-term effect by our manipulation without NSC depletion. Intriguingly, in these experiments quiescent NSC also seemed to appear more frequently as doublets in 4D+ than in 4D− mice (Fig 2D, arrowheads) suggesting that they were the result of an increase in symmetric, relative to asymmetric, proliferative divisions, both of which are known to occur in the mouse SVZ (Calzolari et al, 2015). To investigate this, we took advantage of the fact that while 4D-RFP mRNA expression was terminated soon after Dox removal (Fig EV1A’), RFP as a protein persisted over 1 month later (Fig 2D). Hence, we ranked the proportion of BrdU+RFP+ cells that appeared as doublets (nuclei within 10-μm distance) along the SVZ as a proxy for symmetric divisions, revealing a 30% increase in 4D+ mice as compared to 4D− (from 51.7 ± 1.7 to 67.5 ± 5.1%, P < 0.05; Fig 2D) and consistent with the notion that symmetric proliferative divisions underlie the expansion of NSC and their increase in number (Fig 1D). Third, regarding lineage commitment and multipotency of 4D-expanded NSC, we quantified the proportion of BrdU long-term retaining cells expressing the gliogenic marker S100β and found a similar proportion of mature astrocytes in 4D− and 4D+ (43.2 ± 6.5 and 48.0 ± 4.6%, respectively, P = 0.57; Fig 2C; pie graphs). Hence, as a result of the increased number of NSC and similar proportion of gliogenic commitment, the number of mature astrocytes was also increased in 4D+ mice by twofold (1.5 ± 0.1 × 104 vs. 3.6 ± 0.8 × 104 BrdU+S100β+ cells/mm3 in 4D− vs. 4D+, respectively, P < 0.05; Fig 2C; bar graphs). Similar differences were also found after the 60-day chase (0.9 ± 0.1 × 104 vs. 1.3 ± 0.2 × 104 BrdU+S100β+ cells/mm3 in 4D− vs. 4D+, respectively, P = 0.07; Fig EV2B). Finally, we investigated whether the transitorily expanded pool of NSC was capable of undergoing physiological neurogenesis upon silencing of the 4D cassette. Consistent with this, CLARITY treated, whole-mount immunolabeling of 4D+ brains showed widespread distribution of RFP+ cells throughout the entire OB (Movie EV1). Then, we quantified BrdU+ neurons in the OB birthdated during 4D overexpression. In adult mice, most of the newly generated neurons migrating to the OB integrate in the granule cell layer as NeuN+, GABAergic granule cells (Bagley et al, 2007; Imayoshi et al, 2008; Fig 2E). Additionally, a smaller proportion of GABAergic, glutamatergic or dopaminergic periglomerular interneurons is added to the glomerular layer that can be classified in three mutually exclusive populations of calretinin+ (CalR), calbindin+ (CalB), or tyrosine hydroxylase+ (TH) cells (Hack et al, 2005; Parrish-Aungst et al, 2007; Brill et al, 2009). We observed an evident increase in BrdU+ neurons in 4D+ mice (Fig 2E) that contributed to the granule cell and glomerular layers in proportions similar to that observed in 4D− mice (82.6 ± 1.3 and 17.4 ± 1.3% vs. 82.8 ± 2.2 and 17.2 ± 2.2%, for granule cell and glomerular layers, in 4D+ and 4D− mice, respectively, P = 1.0; Fig 2E; pie graphs). Regarding absolute numbers, 30 days after 4D overexpression we observed a similar increase in all neuronal types that reached our threshold of statistical significance for both NeuN+ granule cells (from 4.42 ± 0.46 × 104 to 6.05 ± 0.36 × 104 cells/mm3, P < 0.05) and TH+ periglomerular cells (from 0.23 ± 0.03 × 104 to 0.44 ± 0.07 × 104 cells/mm3, P < 0.05; Fig 2E). Similar differences were found after the 60-day chase, time at which also the increase in CalB+ cells reached statistical significance (Fig EV2C). Together, these data outline the effects of 4D on NSC resulting in their long-term expansion and increased neurogenesis without affecting their multipotency or compensatory effects due to depletion and/or neuronal death. 4D expression in NSC does not alter the morphology or activity of supernumerary neurons We next assessed whether the integration and activity of supernumerary neurons was altered by the nature of our manipulation in progenitor cells. To this aim, we focussed on granule cells since they represent the most abundant type of adult born neurons playing key roles in olfactory discrimination (Abraham et al, 2010; Alonso et al, 2012; Gschwend et al, 2015; Nunes & Kuner, 2015). NSC expansion was induced for 4 days and granule cells in the OB analyzed 30 days later (Fig 3A), time at which adult born neurons are known to be morphologically mature and integrated (Petreanu & Alvarez-Buylla, 2002). Here, in contrast to our previous quantifications of cell numbers alone (Figs 1 and 2), we needed a system that would allow us to identify supernumerary RFP+ neurons in 4D+ mice and compare them with physiologically generated, RFP− newborn neurons of an equivalent age in 4D− or even within 4D+ mice. To mark such age-matched cohort of newborn neurons in 4D− and 4D+ mice, we then crossed the homozygous 4D− and 4D+ lines with RCEGFP-flox+/+ mice (Miyoshi et al, 2010), thus, labeling nestinCreERt2+ NSC upon Tam administration by GFP, irrespective from the presence or absence of RFP. We then compared superficial granule neurons derived from 4D+ NSC (RFP+GFP+) with physiologically generated neurons of the equivalent age but derived from 4D− NSC (RFP−GFP+). Figure 3. Integration and electrophysiological properties of 4D-derived granule cells A. Experimental design to assess the integration of 4D-derived neurons. B. Fluorescence pictures (left) of immunolabeled GFP+RFP− (4D−) and GFP+RFP+ (4D+) apical dendrites of superficial granule neurons and quantifications (middle and right) of spine density and total dendritic length (box and whiskers) and 3D-Sholl (line graph profile) of apical dendrites starting from the soma (drawings) as the mean number of intersections at 10-μm intervals. C. 3D reconstruction of multi-channel confocal" @default.
• W2908799316 created "2019-01-25" @default.
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• W2908799316 title "An increase in neural stem cells and olfactory bulb adult neurogenesis improves discrimination of highly similar odorants" @default.
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